Gene conversion in the chicken immunoglobulin locus: a paradigm of homologous recombination in higher eukaryotes

Gene conversion was first defined in yeast as a type of homologous recombination in which the donor sequence does not change. In chicken B cells, gene conversion builds the antigen receptor repertoire by introducing sequence diversity into the immunoglobulin genes. Immunoglobulin gene conversion continues at high frequency in an avian leukosis virus induced chicken B cell line. This cell line can be modified by homologous integration of transfected DNA constructs offering a model system for studying gene conversion in higher eukaryotes. In search for genes which might participate in chicken immunoglobulin gene conversion, we have identified chicken counterparts of the yeast RAD51, RAD52, and RAD54 genes. Disruption and overexpression of these genes in the chicken B cell line may clarify their role in gene conversion and gene targeting.     

The effect of growth hormone replacement on cortisol metabolism and glucocorticoid sensitivity in hypopituitary adults

OBJECTIVE: Growth hormone (GH) replacement therapy in hypopituitary adults is associated with sodium and water retention. The underlying mechanisms are incompletely understood and a possible contribution of altered cortisol metabolism or action has not been evaluated. We have investigated the effect of GH replacement therapy on cortisol metabolism, cortisol binding globulin and in-vitro glucocorticoid sensitivity in a group of adult hypopituitary patients. DESIGN AND PATIENTS: We studied 19 adult hypopituitary patients (18 adult onset, M:F, 6:13), who were receiving conventional hydrocortisone (16 patients), thyroxine (14 patients), triiodothyronine (1 patient), sex steroid (9 patients), human chorionic gonadotrophin (1 patient) or desmopressin (6 patients) replacement during a 6-month, double blind controlled trial of GH therapy (active:placebo, 8:11) followed by a 6-month open phase of GH (mean dose: 0.2 IU/kg/week, range 0.051-0.27) and after a 6-week washout phase following discontinuation of GH therapy. MEASUREMENTS: Twenty-four-hour urine free cortisol, cortisol metabolites (CoM), ratio 11-hydroxy/11-oxo CoM (F/E) and ratio 5 alpha/beta tetrahydrocortisol were measured at 6 months, 12 months and after the 6 week washout phase. Serum cortisol binding globulin was measured basally, at 6 months, 12 months and after washout. Glucocorticoid sensitivity was determined in lymphocyte preparations from 8 patients, during GH therapy and after washout, using an in-vitro technique dependent on dexamethasone suppression of phytohaemagglutinin-stimulated thymidine incorporation into DNA. Plasma renin activity and aldosterone were measured after 6-12 months GH therapy and after washout. RESULTS: After 6 months of GH, in patients on hydrocortisone (n = 9), there were significant decreases in CoM (mean decrement 21%, P < 0.01), F/E (mean decreased from 1.27 to 1.0, P = 0.04; reference range 0.33-1.29) and 5 alpha/5 beta tetrahydrocortisol (mean decreased from 0.67 to 0.48, P = 0.01) and a subsequent increase after washout. Patients not on hydrocortisone (n = 2) demonstrated a normal basal F/E falling by 25% on GH therapy but no change in CoM. During 12 months of GH therapy, patients on hydrocortisone (n = 7) demonstrated a further trend to decrement in CoM (P = 0.09) which reversed after washout (P = 0.04). Urine free cortisol tended to fall during GH therapy and increased significantly following washout after 12 months treatment (P < 0.02). Serum cortisol binding globulin decreased by 20% (P < 0.05) during 12 months GH treatment but remained within the reference range. In-vitro studies demonstrated a trend to reduced glucocorticoid sensitivity on GH therapy; the maximum inhibition of phytohaemagglutinin by dexamethasone tended to be less on GH therapy (P = 0.052) and was also lower than in 29 normal volunteers (P < 0.05). There were no significant changes in plasma renin but there was a small increment in aldosterone in recumbent patients (P = 0.04) during the open phase of GH therapy in the placebo arm. CONCLUSIONS: GH therapy in hypopituitary adults is associated with an apparent reduction in availability of administered hydrocortisone as measured by urine cortisol metabolites and urine free cortisol. This effect is unlikely to be clinically significant except possibly in ACTH deficient subjects on suboptimal hydrocortisone replacement. The changes in F/E suggest that GH may directly or indirectly modulate the activity of 11 beta-hydroxysteroid dehydrogenase. The apparent decrease in glucocorticoid sensitivity during GH therapy, demonstrated in vitro, merits further investigation.     

Skin disease affecting the conservation of the western swamp tortoise (Pseudemydura umbrina)

OBJECTIVE: To review the present position of the western swamp tortoise (Pseudemydura umbrina) as an endangered species and significant health issues affecting efforts to save it from extinction. PROCEDURE: A retrospective analysis of the husbandry, hospital and pathology records of the western swamp tortoise captive breeding program at Perth Zoo. RESULTS: In 1987 a captive breeding project was developed to prevent the extinction of the western swamp tortoise but an outbreak of a necrotising dermatitis in 1989 threatened the survival of the captive bred hatchlings. Less severe outbreaks occurred in 1990 and 1993, with isolated cases in between. Of 283 tortoises that were born in captivity or came into captivity from the wild, 37 (13.1%) were affected, comprising 37% of all males, 26% of all females and 13% of animals of unknown gender. Of the affected animals, 70% were less than 2 years of age and 29% were older. Males were 1.6 times more likely to be infected than females but this difference was not statistically significant (P = 0.27). Culture of the lesions consistently yielded unidentified Pseudomonas sp. CONCLUSION: Improved husbandry, such as strict maintenance of water quality and temperature conditions similar to that of the animal's natural habitat, and monitoring the health of individual tortoises have successfully controlled skin disease in the captive breeding of the western swamp tortoise.     

Monitoring of lineage-specific chimaerism allows early prediction of response following donor lymphocyte infusions for relapsed chronic myeloid leukaemia

Thrombogenesis in the left atrial appendage (LAA) has been related to the special morphology of this cavity and to its size and degree of dysfunction. However, no study has focused on LAA function in conjunction with left atrial (LA) function in both sinus rhythm (SR) and nonrheumatic idiopathic atrial fibrillation (AF) in relation to clinical status (cardioembolic stroke). Forty-three patients in SR (14 patients with stroke, 29 control subjects) and 45 patients in AF (27 patients with stroke, 18 control subjects) were examined by transthoracic and transesophageal echocardiography. Baseline clinical characteristics and standard transthoracic and transesophageal measurements of the LA and LAA (size, fractional area change, flow measurements, spontaneous echo contrast, and thrombus) were recorded and compared in relation to cardiac rhythm. Patients in the stroke-SR group showed a significant decrease of fractional area change in the LA (32%+/-15%) and LAA (34%+/-15%) in relation to control subjects (43%+/-10%, p = 0.035, 49%+/-13%, p = 0.006, respectively). Patients in the stroke-AF group showed significant reduction of appendage flow measurements (outward velocity = 22+/-13 vs 33+/-19 cm/sec, p = 0.036), whereas no differences were detected in the center of the LA. In multiple regression analysis, the presence of cardioembolic stroke was positively associated with the presence of spontaneous echo contrast (p = 0.0253) and spontaneous echo contrast negatively associated with appendage inward flow velocity (p<0.001). Cardioembolic stroke in patients in SR is associated with a global decrease of shortening in both cavities and in patients with AF, with a reduction of LAA flow parameters. Patients with spontaneous echo contrast, thrombus, or both showed further reduction of shortening and flow velocities in both cavities, indicating a more advanced stage of dysfunction.     

Comparison of bond strengths of three denture base resins to treated nickel-chromium-beryllium alloy

The success of radiotherapy in eradicating tumours depends on the total radiation dose, but what limits this dose is the tolerance of the normal tissues within the treatment volume. Studies involving fibroblast survival have demonstrated the theoretical feasibility of a predictive assay of radiation sensitivity, but such an assay is still far from clinical application. Using pulsed-field gel electrophoresis (PFGE), we have quantified the initial "apparent" number of DNA double-strand breaks (dsb) induced by the radiation as an alternative measure of sensitivity in 2 different normal cell types from the same patients, epidermal skin cells and lymphocytes. We found significant inter-individual variation in the measured dsb (1-5 dsb/Gy/DNA unit). We also found a linear correlation between molecular damage in lymphocytes and skin samples from the same patient (slope = 0.83; r = 0.694; p = 0.0001). These results suggest that the initial number of dsb could be used as an indicator of the in vivo response to radiation.     

The potential of subtype-selective neuronal nicotinic acetylcholine receptor agonists as therapeutic agents

Neuronal nicotinic acetylcholine receptors (NAChRs) are pentameric ligand-gated ion channel receptors which exist as different functional subunit combinations which apparently subserve different physiological functions as indicated by molecular biological and pharmacological techniques. It is possible to design and synthesize novel compounds that have greater selective affinities and efficacies than nicotine for different NAChRs, which should translate into different behavioral profiles and therapeutic potentials. Examples of NAChR agonists studied are nicotine, SIB-1508Y, SIB-1553A and epibatidine. These compounds have different degrees of selectivity for human recombinant NAChRs, different neurotransmitter release profiles in vitro and in vivo and differential behavioral profiles. Preclinical studies suggest that SIB-1508Y is a candidate for the treatment of the motor and cognitive deficits of Parkinson's disease, whereas SIB-1553A appears to have potential as a candidate for the treatment of Alzheimer's disease. Epibatidine has a strong analgesic profile, however the ratio between pharmacological activity and undesirable effects is so low that it is difficult to envisage the use of this compound therapeutically. Nicotine has a broad profile of pharmacological activity, for instance demonstrating activity in models for cognition and analgesia. As for epibatidine, the adverse effects of nicotine severely limits its therapeutic use in humans. The discovery of subtype-selective NAChR agonists such as SIB-1508Y and SIB-1553A provides a new class of neuropsychopharmacological agents with better therapeutic ratios than nonspecific agents such as nicotine.     

Purification, characterization, and spasmogenic activity of neurotensin from the toad Bufo marinus

Neurotensin (NT) was isolated from an extract of the intestine of the cane toad, Bufo marinus and its primary structure established as: pGlu-Ala-Ile-Val-Ser-Lys-Ala-Arg-Arg-Pro-Tyr-Ile-Leu. This amino acid sequence shows five substitutions (Leu2 --> Ala, Tyr3 --> Ile, Glu4 --> Val, Asn5 --> Ser, and Pro7 --> Ala) compared with bovine NT. Synthetic Bufo NT (pD2 = 8.05 +/- 0.28) was equipotent and equally effective as bovine NT (pD2 = 8.24 +/- 0.38) in producing spasmogenic contraction of isolated segments of toad small intestine. However, the maximum response produced by Bufo NT was only 35 +/- 2% of that produced by substance P. The potencies, but not the maximum responses, to Bufo and bovine NT were significantly (p < 0.05) attenuated by pre-treatment with atropine but neither parameter was significantly diminished by tetrodotoxin and indomethacin. The data suggest that the action of NT involves interaction with receptors on toad intestinal smooth muscle that recognize the C-terminal region of NT (residues 8-13) that has been fully conserved during evolution of tetrapods. Contractile activity is mediated, at least in part, by release of acetylcholine.